Apoptosis or programmed cell death eliminates redundant or damaged cells in metazoans. Abnormal apoptosis contributes to the pathogenesis of many human diseases including cancer and ischemic heart and brain injuries. Bcl-2 and Bax function as a suppressor and a promoter of apoptosis, respectively. Aberrant expression of either protein leads to dysregulation of apoptosis or onset of apoptotic diseases. Bcl- 2 is anchored on the mitochondrial outer membrane, while Bax stays in the cytosol in normal healthy cells. During apoptosis induction Bcl-2 maintains normal membrane function, thereby protecting the organelle and hence the cell. In contrast Bax inserts into the mitochondrial membrane and permeabilizes it. Structures of both proteins were determined in solution and are strikingly similar to each other. In synthetic membranes both proteins form pores, but we do not know whether they form pores in the mitochondrial membrane, and if they do, how their pores differentially affect the mitochondrial permeability. Our long-term goal is to find ways to interfere with the apoptosis program and control diseases such as cancer and stroke. Our short- term goal is to determine why Bcl-2 inhibits apoptosis, whereas the structurally similar Bax promotes it. Our hypothesis is that Bcl-2 and Bax form different structures in the mitochondrial membrane in response to BH3-only proteins, thereby differentially regulate the membrane permeability and apoptosis. We will address three specific aims. 1) What is the structure of Bcl-2 in membranes after it interacts with Bax and/or a BH3- only protein? 2) How does Bcl-2 interact with Bax to prevent Bax from forming a cytochrome c-releasing pore? Do BH3-only proteins change the Bcl-2/Bax interaction? 3) How do small chemical Bcl-2 inhibitors inhibit Bcl-2 in membranes? We will generate a series of site-specifically labeled, fluorescent or photoreactive Bcl-2 and Bax proteins, and use fluorescence and photocrosslinking approaches to resolve these important cell death-or-life issues. The results of the project will establish a structural basis for understanding the functions of Bcl-2 and Bax in regulating both membrane permeability and apoptosis. This knowledge is a prerequisite for evaluating any therapeutic agents that target Bcl-2 and Bax for treatment of apoptotic diseases like cancer and cardiac ischemia.